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PKC-induced ERK1/2 interactions and downstream effectors in ovine cerebral arteries

Yu Zhao, Lubo Zhang, Lawrence D. Longo
2005 American Journal of Physiology. Regulatory Integrative and Comparative Physiology  
PKC-induced ERK1/2 interactions and downstream effectors in ovine cerebral arteries. Both protein kinase C (PKC) and extracellular signal-regulated kinases (ERK1/2) are involved in mediating vascular smooth muscle contraction. We tested the hypotheses that in addition to PKC activation of ERK1/2, by negative feedback ERKs modulate PKC-induced contraction, and that their interactions modulate both thick and thin myofilament pathways. In ovine middle cerebral arteries (MCA), we measured isometric
more » ... measured isometric tension and intracellular free calcium concentration ([Ca 2ϩ ]i) responses to PKC stimulation [phorbol 12,13-dibutyrate (PDBu), 3 ϫ 10 Ϫ6 M] in the absence or presence of ERK1/2 inhibition (U-0126, 10 Ϫ5 M). After PDBu Ϯ ERK1/2 inhibition, we also examined by Western immunoblot the levels of total and phosphorylated ERK1/2, caldesmon Ser789 , myosin light chain 20 (MLC 20 ), and CPI-17. PDBu induced significant increase in tension in the absence of increased [Ca 2ϩ ]i. PDBu also increased phosphorylated ERK1/2 levels, a response blocked by U-0126. In turn, U-0126 augmented PDBu-induced contractions. PDBu also was associated with significant increases in phosphorylated caldesmon Ser789 and MLC20 levels, each of which peaked at 5 to 10 min. PDBu also increased phosphorylated CPI-17 levels, which peaked at 2 to 3 min. Rho kinase inhibition (Y-27632, 3 ϫ 10 Ϫ7 M) did not alter PDBu-induced contraction. These results support the idea that PKC activation can increase CPI-17 phosphorylation to decrease myosin light chain phosphatase activity. In turn, this increases MLC 20 phosphorylation in the thick filament pathway and increases Ca 2ϩ sensitivity. In addition, ERK1/ 2-dependent phosphorylation of caldesmon Ser789 was not necessary for PDBu-induced contraction and appears not to be involved in the reversal of caldesmon's inhibitory effect on actin-myosin ATPase. vascular smooth muscle; U-0126; caldesmon; CPI-17; myosin light chain 20
doi:10.1152/ajpregu.00847.2004 pmid:15956760 fatcat:xbfywp3m5bflhom7rmw4awogtm